Field of the Invention
The present invention relates to a phase contrast microscope, specifically, a phase contrast microscope capable of obtaining a high-contrast phase contrast image even in the case where the thickness of a sample is thick.
Description of the Related Art
A phase contrast microscope is a microscope including a ring slit disposed at a pupil position in an illumination optical system (condenser lens) and an annular phase film disposed at a pupil position in an observation optical system (objective), and converting a phase distribution of a sample into an image contrast by making the ring slit and the phase film to be a conjugate relation.
An image formed with the phase contrast microscope is composed of a plurality of image components such as a bright-field component obtained by ordinary bright-field observation of the sample and the like, in addition to an image component proportional to the phase distribution of the sample. Furthermore, the composition ratio of these image components and the like change depending on the thickness and the shape of the sample.
In order to enhance the contrast of the image component proportional to the phase distribution of the sample, it is necessary to make a state such that the conjugate relation between the phase film in the objective and the ring slit in the condenser lens are maintained properly. Moreover, since the contrast of the image component proportional to the phase distribution changes depending on the radius and the film width of the annular phase film disposed in the objective, under the circumstances, it is preferable that the radius of the phase film is designed to be about half the radius of a pupil of the objective.
In an objective used in a typical phase contrast microscope, the radius of the phase film is designed to be about half the radius of the pupil of the objective so that the contrast of the phase contrast image at each magnification is substantially the same as that of another one. When the objective is exchanged for another objective suitable for a desired observation magnification in order to change observation magnification, a projected magnification, which is a magnification in the case where the ring slit in the condenser lens is projected on the pupil position of the objective, changes.
In order to perform the phase contrast observation, it is necessary to maintain the conjugate relation between the phase film and the ring slit, and thus, it is necessary to change the radius of the ring slit depending on the radius of annular phase film disposed in the objective for each magnification. For this reason, it is necessary to prepare ring slits corresponding to the respective magnifications of objectives in advance.
The phase contrast microscope is, for example, used for observing the development of cultured cells. The development of the cultured cells is observed in accordance with a predetermined procedure in which observation magnification and the like are often changed. In such an observation, work efficiency is reduced if a ring slit is changed to another one corresponding to an objective to be used. To address the problem, a microscope capable of phase contrast observation using a common ring slit has been disclosed in Japanese Patent Application Publication Raid-open No. 11-084260.
The phase contrast microscope disclosed in Japanese Patent Application Publication Raid-open No. 11-084260 is configured to enable phase contrast observation using the same ring slit even if magnifications of the objectives to be used are different. In the phase contrast microscope, by making the radius of the common ring slit and the radius of the pupil of each objective to satisfy a certain condition, the change in the contrast of the image component proportional to the phase distribution, which is produced by changing the magnification of the objective, is reduced.
Here, a state in which the position of the sample coincides with the in-focus position of the objective is called a in-focus state (hereinafter referred to as the “in-focus state”), and a state in which the position of the sample does not coincide with the in-focus position of the objective is called a defocus state (hereinafter referred to as the “defocus state”).
When the sample is a colorless and transparent phase object with phase distribution, the contrast of the image component proportional to the phase distribution is not generated under the in-focus state in the bright-field observation of the sample. Therefore, the phase contrast observation or the like is used for observing such a phase object. However, when the sample is moved from the in-focus state to the defocus state (defocused) and is observed under the defocus state, the contrast of the image component proportional to the phase distribution is generated. This image contrast is generated such that the wavefront aberration generated by defocusing acts on the phase distribution of the sample as a phase film of a phase contrast microscope. An image of the phase object generated due to an interaction of the phase distribution and the wavefront aberration by defocusing is called an interaction image (hereinafter referred to as the “interaction image”).
A microscope used for observing cultured cells using such an interaction image has been disclosed in Japanese Patent Application Publication Raid-open No. 2005-173288.
An image of the sample formed by the phase contrast microscope is composed of a phase contrast image component proportional to the phase distribution of the sample and an image component obtained by observing a phase object in bright-field observation is described. In the case where the sample is placed in the defocus state, since the interaction image overlaps with the image component proportional to the phase distribution, contrast of the phase contrast image decreases.
Moreover, since the sample is placed in the defocus state, the image component proportional to the phase distribution also becomes a phase contrast image having blur due to an affection of defocusing. In this case, since a contrast of the image component proportional to the phase distribution decreases, contrast of the image observed by the phase contrast microscope further decreases.
As described above, the cultured cells are available as an observation object for the phase contrast microscope. Conventional cultured cells are cells in which the thickness is thin (hereinafter referred to as the “thin cells”). At the thin cells, if the top surface of the cells is focused, the entire cells from the top surface to the bottom surface are in a substantially in-focus state. At the thin cells, the interaction image is not generated.
As just described, at the thin cells, a formed image is composed of only the image component proportional to the phase distribution at any position from the top surface to the bottom surface. Therefore, in the case where the thickness of the sample is thin, a high-contrast phase contrast image can be obtained in the phase contrast microscope disclosed in Japanese Patent Application Publication Raid-open No. 11-084260.